403© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved.
404 The Immunoassay Handbook
TABLE 1 An A–Z of Sources of Interferences in Immunoassays
(Boscato et al., 1989; Boscato and...
405CHAPTER 5.3 Interferences in Immunoassay
CK-MB values (range 10–1000µg/L in 1008 blood donors)
to <3µg/L following trea...
406 The Immunoassay Handbook
anti-mouse antibodies (HAMA) in the sample” (Services
FDA US DoH, 1996). A subsequent FDA rec...
407CHAPTER 5.3 Interferences in Immunoassay
Paraproteins and Immune Complexes
The presence of paraproteins has been associ...
408 The Immunoassay Handbook
Elevated levels of bilirubin ...
409CHAPTER 5.3 Interferences in Immunoassay
For example in the case of hCG assays, the FDA has rec-
ommended that “specific...
410 The Immunoassay Handbook
intensive chart review or laboratory investigation could
be conducted.
Another powerful tool ...
411CHAPTER 5.3 Interferences in Immunoassay
example, with β-hCG, the serum test is susceptible to
interference, but the ur...
412 The Immunoassay Handbook
laboratory; this was consistent with ectopic pregnancy, and
the patient was treated with low-...
413CHAPTER 5.3 Interferences in Immunoassay
seminomatous GCT who had a falsely elevated hCG at his
routine 5-year follow-u...
414 The Immunoassay Handbook
the conjugation of the anti-tacrolimus monoclonal anti-
body to the beta-galactosidase report...
415CHAPTER 5.3 Interferences in Immunoassay
Frodin, J.E., Lefvert, A.K. and Mellstedt, H. The clinical significance of ham...
416 The Immunoassay Handbook
Owen, W.E., Rawlins, M.L. and Roberts, W.L. Selected performance characteris-
tics of the Roc...
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The immuassay handbook parte43

  1. 1. 403© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/B978-0-08-097037-0.00027-0 Immunoassays are a critical high-sensitivity detection technology in modern clinical laboratories, but their Achilles’ heel is their susceptibility to interferences. The presence of interfering substances in a patient’s sample can cause an erroneous test result, either false positive or false negative. This testing error can be clinically signifi- cant and may lead to misdiagnosis and disastrous conse- quences for the patient. There have been extensive reviews of interferences in immunoassays (Boscato et al., 1989; Boscato and Stuart, 1986, 1988; Braunstein, 2002; Diamandis, 2004; Ismail and Barth, 2001; Ismail et al., 2002b; Itoh and Yamaguchi, 1995; Jones, 2002; Kohse and Wisser, 1990; Kricka, 1999, 2000; Kricka et al., 1990; Kroll and Elin, 1994; Levinson, 1992; Tate and Ward, 2004; Van Kroonenburgh and Pauwels, 1988; Weber et al., 1990), and here, we present an overview of the scope of immunoassay interferences, methods to combat analytical problems attributable to their presence in bio- logical fluids, and clinically significant examples. Immu- noassay interferences are a long-standing problem that date back to serologic tests in the early 1900s (Page and Heimoff, 1946; Seelman, 1918). As one author stated in correspondence about Wassermann testing for syphilis (Seelman, 1918): “There is only one thing I am more certain of than this, outside of death and taxes, that if I obtained a positive reaction on my blood in a test made by the method described, I would not accept the verdict as final, but would proceed to make another more accurate and reliable test.” In the modern era, reports in the early 1970s described false-positive interferences in immunoassays for hepatitis B surface antigen due to circulating antibodies (Hollinger, 1972; Prince et al., 1973). Nearly 40 years after this publica- tion, immunoassay interference is still a problem, but countermeasures have become more sophisticated, and awareness among laboratorians and the general public has increased significantly. The complicating factors of many types of interferences are that the effect may vary from method to method, from test to test, from patient to patient, and it may also vary with time—all of which con- tribute to making this critical issue difficult to solve. Scope of Immunoassay Interferences Table 1 summarizes the principal sources of interferences in immunoassays. Interferences can arise in the preanalyti- cal and analytical phase of an assay, and these are briefly described in the following sections. CIRCULATING ANTIBODIES Anti-animal Antibodies An anti-animal antibody can lead to a positive or, less com- monly, a negative interference in a sandwich or two-site immunoassay. A positive interference arises as a result of the anti-animal antibody bridging the animal-derived capture antibody and antibody conjugate as depicted in Fig. 1B. The net result is binding of the conjugate that is indistin- guishable from the binding of the conjugate that occurs in the presence of the specific antigen. A negative interference is thought to be due to the interfering anti-animal antibody binding to either the capture antibody or the antibody con- jugate such that bridging of these antibodies by analyte (sandwich formation) is inhibited (Fig. 1C). A circulating anti-animal antibody often arises as a result of the normal response of the immune system to the admin- istered “foreign” protein antigen (e.g., murine monoclonal IgG). An increasing number of therapeutic and diagnostic pharmaceutical agents are derived from animal sources or are affinity purified using immobilized monoclonal mouse antibodies that may detach and co-purify with the protein and as such provide the basis of an immunological challenge (Smid and van der Meer, 1995). Some unconventional therapies intended as tonics also contain antigenic animal proteins, such as “antireticulocytoxique” (lyophilized serum obtained from rabbits injected with homogenates of human bone marrow and spleen) (Schaison et al., 1981). Vaccina- tion against infectious diseases (Schaison et al., 1981; Arnold et al., 1994; Padova et al., 1991) and blood transfusion (Hawkins et al., 1980) are other possible routes by which animal protein antigens may trigger antibody formation. Indeed, some vaccines have residual protein from chick embryo or egg cultures, or rabbit serum (Schaison et al., 1981; Padova et al., 1991); although, the role of influenza vaccination in false-positive viral antibody results has been questioned (Simonsen et al., 1995). Tables 2 and 3 list the examples of diagnostic and pharmaceutical agents based on animal or recombinant proteins (Elbakri et al., 2010). There are also non-iatrogenic causes of anti-animal antibodies, and these include keeping pets (Berglund and Holmberg, 1989), placental transfer to the unborn child (Czernichow et al., 1981; Larsson et al., 1981), animal husbandry, and the transfer of dietary antigens across the gut (Falchuk and Isselbacher, 1976; Jewell and Truelove, 1972). Human anti-mouse antibodies (HAMAs) have been the most troublesome antibodies and hence the focus of numer- ous investigations. Several studies have sought to deter- mine the prevalence of HAMA or of interfering antibodies generally, and estimates vary widely. One study reported a prevalence of ~9% based on the reduction of apparent Interferences in Immunoassay Jason Y. Park (jason.park@childrens.com) Larry J. Kricka (kricka@mail.med.upenn.edu) C H A P T E R 5.3
  2. 2. 404 The Immunoassay Handbook TABLE 1 An A–Z of Sources of Interferences in Immunoassays (Boscato et al., 1989; Boscato and Stuart, 1986, 1988; Braunstein, 2002; Diamandis, 2004; Ismail and Barth, 2001; Ismail et al., 2002b; Itoh and Yamaguchi, 1995; Jones, 2002; Kohse and Wisser, 1990; Kricka, 1999, 2000; Kricka et al., 1990; Kroll and Elin, 1994; Levinson, 1992; Tate and Ward, 2004; Van Kroonenburgh and Pauwels, 1988; Weber et al., 1990) Anti-analyte antibodies Anti-animal antibodies (cow, goat, horse, mouse, pig, rabbit, sheep, rat) Autoantibodies Blood substitutes Carryover Collection tube Contamination Contrast agents Complement Cross-reactivity Drugs Drug metabolites Fibrin Hemolysis Herbal remedies Heterophile antibodies High-dose hook effect Human anti-animal antibodies Imaging agents Immune complexes Insufficient sample volume Lipemia Microclots Paraproteins Partially filled collection tubes RF Sample storage Sample matrix FIGURE 1 Sandwich immunoassay for a true antigen-positive (a) and the mechanism of false-positive (b) and false-negative (c) interference. TABLE 2 Pharmaceutical and Experimental Drug Agents Derived from Animal Sources Animal Source Agent Chicken Hyaluronic acid Cow Insulin Horse Anti-thymocyte globulin, Premarin Malayan pit viper Ancrod Mouse Monoclonal antibody therapeutic and imaging agents Pig Factor VIII, insulin, heparin Rat Monoclonal antibody therapeutic agents Salmon Calcitonin Sheep Digibind™ TABLE 3 Therapeutic Monoclonal Antibodies Antibody (Brand Name) Treatment Indication (Target) Murine antibodies (suffix—omab) Ibritumomab tiuxetan (Zevalin) Non-Hodgkin lymphoma (CD20) Muromonab-CD3 (Orthoclone OKT3) Transplant rejection (T cell CD3 Receptor) Tositumomab (Bexxar) Non-Hodgkin lymphoma (CD20) Chimeric Mouse/human (suffixes—ximab) Abciximab (ReoPro) Cardiovascular disease (glycoprotein IIb/IIIa) Cetuximab (Erbitux) Colorectal cancer, head and neck cancer (epidermal growth factor receptor) Infliximab (Remicade) Autoimmune disorders (TNF-α signaling) Rituximab (Rituxan, Mabthera) Non-Hodgkin lymphoma (CD20) Basiliximab (Simulect) Transplant rejection (CD25) Humanized antibodies from mouse (suffix—zumab) Bevacizumab (Avastin) Colorectal cancer, age-related macular degeneration (vascular endothelial growth factor) Certolizumab pegol (Cimzia) Crohn’s disease (TNF-α signaling) Daclizumab (Zenapax) Transplant rejection Eculizumab (Soliris) Paroxysmal nocturnal hemoglobinuria (C5) Efalizumab (Raptiva) Psoriasis (CD11a) Gemtuzumab (Mylotarg) Acute myelogenous leukemia (CD33) Natalizumab (Tysabri) Multiple sclerosis and Crohn’s disease (alpha-4 integrin) Omalizumab (Xolair) Mainly allergy-related asthma (IgE) Palivizumab (Synagis) Respiratory syncytial virus (RSV F protein) Trastuzumab (Herceptin) Breast cancer Ranibizumab (Lucentis) Macular degeneration (vascular endothelial growth factor A) Humanized antibodies from rat Alemtuzumab (Campath) Chronic lymphocytic leukemia (CD52) Rat–murine hybrid Ertumaxomab (Rexomun) Breast cancers (CD3E)
  3. 3. 405CHAPTER 5.3 Interferences in Immunoassay CK-MB values (range 10–1000µg/L in 1008 blood donors) to <3µg/L following treatment with nonimmune mouse serum (Thompson et al., 1986). Another study demon- strated interfering antibodies in a set of 40 samples evalu- ated by four immunoassays (thyroid-stimulating hormone (TSH), prostate-specific antigen (PSA), β-human chori- onic gonadotropin (β-hCG), and cortisol) (Emerson et al., 2003). Three different interference assessment methods revealed an interfering antibody in 7.5–16.2% of samples tested in a competitive immunoassay (cortisol) and 0–83.6% of samples tested in sandwich immunoassays (Table 4). However, less than 11% of the test interferences were clin- ically significant (changing the result into or out of the ref- erence interval). Based on heterophile blocking reagent (HBR) criterion, only 2.9% of tests had interference. Based on serial dilution criterion, only 10.8% of tests had interfer- ence. A large study involving testing for TSH and gonado- tropins found a significant interference in samples from 28 of the 5310 patients tested (Ismail et al., 2002a). Another broad based investigation examined results on samples from 10 donors tested for 74 analytes in 66 laboratories in 10 countries and found that 8.7% of the 3445 results were false positives due to interference (Marks, 2002). Human anti-animal antibody responses can be of differ- ent classes (IgG, IgA, IgM, or rarely IgE) (Chatenoud et al., 1986; Frodin et al., 1992; Iitaka et al., 1991; McCarthy et al., 1988), and they can have anti-idiotype, anti-isotype, or anti-anti-idiotype specificity (Frodin et al., 1992; Reins- berg, 1995). Generally, isotype antibodies are more com- mon than idiotype antibodies (Lind et al., 1991), but some instances of exclusively anti-idiotype antibodies have been reported (Legouffe et al., 1994). The magnitude and dura- tion of a HAMA response show great variability. The reported serum concentrations range from µg/L to g/L (Ledermann et al., 1988; Moseley et al., 1988) and can per- sist in blood from weeks to months after exposure to mouse immunoglobulin (Baum et al., 1994; Sharma et al., 1992). Circulating antibodies with specificities for a range of animal immunoglobulins have been reported; anti-rabbit (IgG, IgA, and IgM) (Hiemstra et al., 1988) and anti-goat antibodies (Larsson et al., 1981; Vandalem et al., 1980) are particularly important because these animals are sources of antisera for immunoassay reagents. For example, human anti-rabbit antibodies (HARA) and human anti-horse anti- bodies can arise following treatment with the immunosup- pressant agents, rabbit and equine anti-thymocyte globulin (ATG), respectively (Hiemstra et al., 1988; Harkiss, 1984). Other anti-animal antibodies causing interferences include anti-sheep antibody (Hunter and Budd, 1980) and anti-cow antibody (Kwong and Teale, 1994). In addition, human anti-chimeric antibodies have been detected in patients treated with chimeric antibodies (Afif et al., 2010; Buist et al., 1995; Goto et al., 2009). Not all animal-derived agents lead to antibody produc- tion. Administration of Digibind™, a sheep anti-digoxin-Fab used to treat digoxin poisoning, has not been reported to lead to the formation of anti-sheep antibodies. Also, not all antibodies cause interferences. Human anti-porcine anti- bodies are formed in hemophiliacs treated with porcine fac- tor VIII, but no assay interferences have been reported (Morrison et al., 1993). Another issue is that sequence homology between IgG molecules from different animal species can lead to cross- reactivity between anti-animal antibodies and animal immunoglobulins. This has been illustrated by a study in which interfering antibodies could be blocked with similar efficacy by mouse IgG1, mouse IgG2a, rat IgG and by mouse, goat, or sheep serum (Sampson et al., 1994). Another study showed that positive interferences in a two- site CK-MB assay could be reduced by 80% using nonim- mune animal sera in the order of effectiveness of mouse> sheep=cow>rat>guinea pig>rabbit cat=dog (pigeon sera had no effect) (Thompson et al., 1986). The FDA has recognized the problems that can arise from anti-animal antibodies and requires that the package insert of an in vitro diagnostic device state the following as a limitation: “As with any assay employing mouse anti- bodies, the possibility exists for interference by human TABLE 4 Screening for Immunoassay Interferences Using Three Methods (Emerson et al., 2003) Interference Test ICON HBR Pretreatment Serial Dilutions Definition of interference Positive color reaction on negative control zone Significant discrepancy between result on a 1:1 dilution before and after HBR treatment Significant discrepancy between result on a 1:1 dilution and any other dilution Competitive immunoassay Test % Positive % Positive % Positive Cortisol (polyclonal goat anti-rabbit capture and polyclonal rabbit conjugate) 7.5% 11.1% 16.2% Sandwich immunoassay Test % Positive % Positive % Positive TSH (polyclonal goat anti-mouse: monoclonal mouse capture and polyclonal goat conjugate) 5% 55.6% 38.2% PSA (monoclonal mouse capture and monoclonal mouse conjugate) 0% 17.5% 55% β-hCG (polyclonal goat anti-mouse: monoclonal mouse capture and polyclonal rabbit conjugate) 0% 73% 83.8%
  4. 4. 406 The Immunoassay Handbook anti-mouse antibodies (HAMA) in the sample” (Services FDA US DoH, 1996). A subsequent FDA recommenda- tion stated: “If the assay kit employs mouse monoclonal antibodies, include a warning that specimens from patients who have received preparations of mouse monoclonal antibodies for diagnosis or therapy may contain human anti-mouse antibodies (HAMA) and may show either falsely elevated or depressed values when tested” (Services FDA US DoH, 1996). Circulating anti-animal antibodies are not just a prob- lem for humans. In the veterinary world, canine anti- mouse antibodies (CAMAs) were found after treatment with mouse monoclonal agents (e.g., mouse monoclonal antibody 231 for lymphoma), and low levels of preexisting CAMA were found in all of the dogs tested (Jeglum, 2009). False-positive test results have also been seen in tests designed to detect feline leukemia virus infection in cats; this was attributed to the presence of circulating antibodies directed against mouse immunoglobulins. This study esti- mated that 0.14–0.57% of cats had anti-mouse antibodies (Lopez and Jacobson, 1989). Anti-microorganism Antibodies Antibodies to microorganisms can also be a source of interference. Escherichia coli septicemia, in a patient with a restricted IgM lambda paraprotein, has been associated with increased false-positive immunoassay test results for cardiac troponin I, thyrotropin, hCG, alpha-fetoprotein, and CA-125. The falsely positive results could be normal- ized (and the IgM lambda paraprotein removed) by incu- bation with irrelevant murine monoclonal antibodies or with formalin-killed E. coli from the patient’s infection, thus indicating that the IgM lambda antibody response produced an antibody that had anti-immunoglobulin activity and caused the falsely increased assay results (Covinsky et al., 2000). Heterophile Antibodies Heterophile antibodies are antibodies produced against poorly defined antigens, and these are generally weak anti- bodies with multi-specific activities. This is in contrast to strong human anti-animal antibodies produced against well-defined antigens as a result of treatment with animal immunoglobulins (Kaplan and Levinson, 1999). Hetero- phile antibodies can bridge capture and conjugate antibod- ies in sandwich assays but cannot compete well with the high-affinity antigens in competitive binding assays. It has been suggested that antibodies should be called hetero- phile when: “there is no history of medicinal treatment with animal immunoglobulins or other well-defined immu- nogens and the interfering antibodies can be shown to be multi-specific (react with immunoglobulins from two or more species) or exhibit natural rheumatoid factor activity” (Kaplan and Levinson, 1999). Antibodies to Assay Reagents The label in an enzyme conjugate can also be the target for circulating antibodies. The observation of an interference associated only with assay systems which employed horse- radish peroxidase but not 125I as a label was suggestive of specificity of the interferent for the peroxidase label (John et al., 1989). Circulating antibodies that recognize an epit- ope present only on the monoclonal antibody–enzyme conjugate have been implicated in a positive interference in a whole-blood tacrolimus immunoassay. Immunoab- sorption studies showed that the interferent bound to the conjugate of antibody and beta-galactosidase but not to the unconjugated antibody or beta-galactosidase (Parikh et al., 2010). Likewise, the ruthenium (Ru) chelate label in an electro- chemiluminescent immunoassay can be the target of inter- fering antibodies. Anti-Ru antibodies have been found in some sera that lead to falsely elevated fT3 results (Elecsys® system). The antibody binds to ruthenylated anti-T3 antibody but not the unlabeled antibody. Interest- ingly, this antibody did not interfere with the Elecsys free thyroxine or TSH assays both of which utilize Ru-labeled antibody reagents, and it is thought that the fT3 assay is more prone to interference because it uses low concentra- tion of Ru-labeled antibodies (Sapin et al., 2007). In the context of electrochemiluminescent assays, interference possibly due to a circulating anti-streptavidin antibody has been thought to be the cause of falsely high serum vitamin D results (Khieng and Stevens, 2010). Autoantibodies and Anti-analyte Antibodies Rheumatoid factor (RF) is a circulating IgM autoantibody found in the serum of most patients with rheumatoid arthritis and in other related and unrelated diseases. It is a known interferent in immunoassays (Despres and Grant, 1998; Hamilton, 1989; Larsson et al., 1991) and in a sand- wich assay interferes by interacting with the Fc region of IgG reagent antibodies. RF can cause false-negative inter- ference by blocking the capture antibody or can cause false-positive interference by bridging capture antibody and antibody conjugate (Larsson et al., 1991; Selby, 1999). In one study, positive interference by RF in a cardiac tro- ponin I microparticle enzyme immunoassay was elimi- nated by pretreatment with polyclonal antiserum against RF (Dasgupta et al., 1999a). Anti-insulin antibodies can cause falsely low values of insulin by binding insulin such that it is not able to bind to the assay antibody reagents (Kim et al., 2011), however, removal of such antibodies by precipitation can eliminate this type of interference (Nishii et al., 2010). Various studies have demonstrated the presence of anti- troponin antibodies in different patient groups, including the coprevalence of autoantibodies to cTnI and cTnT in blood donors (Adamczyk et al., 2010; Nussinovitch and Shoenfeld, 2010; Ruchala et al., 2007). These antibodies masked the release of troponin and thus led to false nega- tive test results (Eriksson et al., 2005). Alternatively, persis- tently elevated levels of cTnI have been reported in a patient with cardiac disease, and these were ascribed to a circulating complex of cTnI and IgG (Plebani et al., 2002). Macroprolactin is a complex of prolactin and anti- prolactin, and this circulating complex can cause apparent hyperprolactinemia. However, macroprolactin interfer- ence can be eliminated in prolactin immunoassays by either ultracentrifugation or polyethylene glycol (PEG) precipitation (Beltran et al., 2008; Quinn et al., 2006).
  5. 5. 407CHAPTER 5.3 Interferences in Immunoassay Paraproteins and Immune Complexes The presence of paraproteins has been associated with immunoassay interferences. For example, elevated serum TSH has been reported in a patient with paraproteins (IgG kappa and IgM lambda) that could not be blocked in a heterophile antibody blocking tube. The interference was specific to the UniCel® DxC 880i (no interference on the Architect® i2000SR®) and only normalized after the disappearance of the monoclonal bands (Imperiali et al., 2010). Another way in which a paraprotein can cause apparently aberrant hormone levels is by providing addi- tional hormone-binding capacity. Increased total thyrox- ine (T4) and triiodothyronine (T3) have also been reported due to an IgA-lambda-secreting multiple myeloma. The paraprotein bound both T4 and T3 and thus acted as an additional thyroid hormone-binding protein thus increas- ing the serum concentration of these hormones rather than interfering in the radioimmunoassay (RIA) itself (Cissewski et al., 1993). In some cases of paraproteinemia, the concentration of the paraprotein is so high as to significantly increase serum viscosity, and this can compromise the accurate dispensing of the sample. A sample that is hyperviscous due to a poly- clonal gammopathy has also been shown to cause falsely elevated T4 values in RIAs in a method specific manner (Tamagna et al., 1979). Hyperviscous samples due to circulating immune com- plexes can interfere in nephelometric methods (e.g., IgM, IgA) as a result of precipitation of the complexes by PEG in the reaction mixture. Interference was also found in radial immunodiffusion methods because of failure of immunoglobulins to migrate as a result of molecular inter- actions (Levinson et al., 1988). DRUGS, HERBAL REMEDIES, BLOOD SUBSTITUTES, AND IMAGING AGENTS Pharmaceutical preparations administered to patients for therapeutic or diagnostic purposes (e.g., antibody-based imaging agents) can be the source of a wide range of test interferences including interferences in immunoassays. Drugs A drug may alter the concentration of an analyte by an in vivo action, and this may be the desired or undesired effect of the drug (in vivo interference). Much more com- monly encountered is an in vitro interference ascribable to an effect of a drug or its metabolite(s) on an analytical reac- tion. The panoply of drug interferences is cataloged (Young, 2000). Some drugs cross-react with antibody reagents, e.g., fludrocortisone derivatives cross-react to produce false- positive cortisol results (Berthod and Rey, 1988), in others, a metabolite cross-reacts and complicates measurement of the drug as in the case of cyclosporin A metabolites in immunoassays for cyclosporin A (Steimer, 1999). Other drugs, such as dipyrone have also been shown to interfere in immunoassays that use a peroxidase label (Gascon- Roche et al., 1995). An interesting example of drug inter- ference is provided by Digibind, a digoxin-Fab antibody used in the treatment of digoxin overdose. In vitro it has been shown to cause a negative interference in the mea- surement of total digitoxin concentrations by both fluores- cence polarization immunoassays and to a greater extent in chemiluminescent immunoassays via direct binding to digitoxin (Digibind neutralized both digitoxin and digi- toxigenin in vitro) (Dasgupta et al., 1999b). Herbal Remedies A more recent concern has been interferences caused by herbal remedies, especially in digoxin immunoassays, which can be influenced by the Chinese medicines Chan Su or Dan Shen (Dasgupta and Bernard, 2006). Ingestion of St John’s wort is associated with abnormally low levels of cyclosporine, digoxin, and theophylline (Dasgupta, 2003). Contamination of a Chinese medicine with Western drugs (e.g., phenytoin) is yet another source of false-positive results (Dasgupta and Bernard, 2006). Blood Substitutes Blood substitutes based on polymerized hemoglobin or perfluorocarbon emulsions (e.g., Perflubron) are in devel- opment as temporary oxygen carriers. A deleterious con- sequence of the administration of such agents is that serum and plasma samples from patients receiving hemoglobin- based blood substitutes are red in color, whereas those from patients receiving perfluorocarbon agents appear to be lipemic. Although some immunoassays are unaffected by the presence of a polymerized hemoglobin-based blood substitute, others show positive (e.g., AxSym® gentamycin assay) and negative interferences (e.g., Axsym vancomycin and Stratus® CK-MB assay). However, in these studies, no immunoassay interferences were found due to the pres- ence of the perfluorocarbon-based blood substitute (Ma et al., 1997). Studies with the hemoglobin-based oxygen carrier HBOC-201 (glutaraldehyde-polymerized bovine hemo- globin) revealed no significant interferences produced by the presence of this blood substitute (in vitro concentrations—60g/L) in various therapeutic drug immunoassays (Callas et al., 1997). In contrast, studies with Hemospan®, a PEG-conjugated human hemoglobin, showed a positive interference in a cTnI immunoassay (Beckman Access® II method) (Cameron et al., 2009). Contrast Agents In vitro studies with a series of 12 different contrast agents used in coronary angiography revealed false-positive results for all agents tested using an Opus Magnum™ cTnI assay, but only one contrast medium (Lipiodol®; poppy-seed oil) gave a positive result with the ACCESS cTnI assay (Lin et al., 2006). A separate study demonstrated that both iodine-based radiopaque contrast agents (Ioversol 350, Iopamidol 370, Iomeprol 300, Iomeprol 400, Iohexol 300) and gadolinium-based contrast reagents (gadopentetic acid) interfere with immunoradiometric assays for carcino- embryonic antigen (CEA), CA-130, and tissue polypeptide antigen (Watanabe et al., 1998). However, in a study target- ing interference by gadolinium-based magnetic resonance contrast agents, no immunoassay interference was seen on multiple immunoassay analyzers (Proctor et al., 2004).
  6. 6. 408 The Immunoassay Handbook ICTERUS, HEMOLYSIS, LIPEMIA, AND OTHER SAMPLE MATRIX COMPONENTS Elevated levels of bilirubin (40 mg/dL) have been shown to cause a statistically significant decrease in cTnI con- centrations (but not CK-MB assays) measured using microparticle enzyme immunoassays, but the mecha- nism of this interference is unclear (Dasgupta et al., 2001). A negative interference due to hemolysis or elevated hemoglobin has been observed in a cTnT immunoassay. In addition, proteases released from red blood cells during hemolysis contribute to this effect by degrading cTnT, as evidenced by the reduction in the negative interference by a protease inhibitor (pepstatin A) (Sodi et al., 2006). Dilution of the sample usually limits any adverse effects of lipemia, but nevertheless, it has been shown to cause a negative interference at elevated levels (>22.5g/L) in elec- trochemiluminescent immunoassay for testosterone (Owen et al., 2010). Also, lipemia is a source of interference in nephelometric immunoassays (e.g., apolipoprotein B, hap- toglobin assay) (Bossuyt and Blanckaert, 1999; Vander Heiden et al., 1983). Other components of the sample matrix can produce positive interferences. This has been observed for samples spiked with alkaline phosphatase in a Stratus fluorimetric EIA for cTnI (Dasgupta et al., 2001). This type of assay is based on radial diffusion of sample and reagents away from a central application zone that contains the immobilized capture antibody. High levels of endogenous alkaline phosphatase in a sample may not wash away from the cen- tral zone and thus mimic captured alkaline phosphatase conjugate, and this may lead to a false-positive result (Dasgupta et al., 2001). Fibrinogen can also be the cause of assay interferences when using plasma samples, but the interference can be eliminated by thermal coagulation (Allner, 1985). Tiny fibrin strands due to incomplete sepa- ration of serum can interfere with an immunoassay; how- ever,recentrifugationcaneliminatethistypeofinterference (Nosanchuk et al., 1999). Another interesting example of assay interference arises from matrix instability. Positive interference in a phenyt- oin RIA was previously encountered in liquid control sera that had been shipped internationally (>2 weeks shipping time) (Wild, 1982). The interference was due to the release of nonesterified fatty acids in the control sera during trans- port. It was surmised that the nonesterified fatty acids dis- placed phenytoin that nonspecifically bound to serum proteins. Nonesterified fatty acids made more phenytoin available to compete with labeled phenytoin for antibody binding sites; thus resulting in a falsely positive increase in measured phenytoin. SAMPLING, CARRYOVER, AND CONTAMINATION False positives can also arise as a result of contamination or from carryover during sampling on an automatic analyzer. Good laboratory practice usually eliminates contamina- tion of specimens. Generally, laundry systems on immu- noassay analyzers are optimized so that carryover due to successive transfer of residual sample on a dispensing probe is extremely low (e.g., <0.001%) (Matsushita et al., 1996). A further issue specific to the sample type is that the presence of fibrin in serum samples leads to false-positive test results in immunoassays for cTnI concentrations (AxSym® analyzer method) (Kazmierczak et al., 2005; McClennen et al., 2003). BLOOD COLLECTION TUBES A blood collection tube is a complex device that is fabri- cated from multiple components made of different materials that can interact with the components of a specimen or shed interfering material into the specimen. In the past, this has been a particular issue for therapeu- tic drug monitoring due to drug adsorption to the sepa- rator gel (Bowen et al., 2010), also the different preservatives present in collection tubes and storage conditions can impact test results (Tate and Ward, 2004; Bowen et al., 2010; Evans et al., 2001). Likewise, silicon- ized plastic tubes have been shown to cause false-nega- tive results in ACTH RIAs and false-positive results in a C-reactive protein immunoassay (Chang et al., 2003; Galligan et al., 1996). More recently, immunoassay platform-dependent posi- tive interferences in competitive assays, especially total T3, have been identified and attributed in part to a component of serum separator tubes, the organosilicone surfactant Silwet® L-720 (Bowen et al., 2005, 2007). This surfactant was shown to displace capture antibody from the solid sup- port, and thus in an assay, bound label would be lost, and this would mimic high concentrations of the analyte. Indeed, there is a cocktail of surfactants in each collection tube, and there is a potential for each of these additives to interfere with an immunoassay. This particular example of tube additives causing inter- ference is an important reminder that controls for an assay operate at their best when they are treated exactly the same as a patient sample. The standard practice for most labora- tories with automated immunoassays is to have the control reagents stored within the machine where it is directly sampled by the machine when necessary. In this type of process, the control reagents do not come into contact with the collection tubes, which in the case of collection tube additives was a missed opportunity to detect interfer- ence (Kricka et al., 2005). CROSS-REACTIVITY OF ANTIBODY REAGENTS In the early days of immunoassays, some antisera were cross-reactive, and this caused false positives (e.g., lutein- izing hormone [LH] in hCG assays) (Thomas and Segers, 1985). Nowadays, highly specific antibodies with low cross-reactivity predominate; cross-reactivity can still be a problem in some assays due to the presence of structurally similar substances present in the sample (e.g., drugs or drug metabolites, or hormones that share a subunit) (Steimer, 1999; Datta et al., 1996). The problem of cross- reactivity is specifically addressed in immunoassay package inserts in the SPECIFIC PERFORMANCE CHARACTERISTICS— SPECIFICITY section. This section states the cross-reactivity of the assay antibody with relevant candidate interferents.
  7. 7. 409CHAPTER 5.3 Interferences in Immunoassay For example in the case of hCG assays, the FDA has rec- ommended that “specificity studies be performed on speci- mens with high physiological concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH). High levels of LH should not significantly cross-react with the hCG antibody used. Similar studies may also be performed with human placental lactogen (hPL) and human growth hormone (hGH). Spiking of samples may be necessary” (Services FDA US DoH, 1996). HIGH-DOSE HOOK EFFECTS Another source of interference in the form of a false nega- tive is attributable to a “high-dose hook effect.” In this situation, a high concentration of an analyte gives similar response to that of a much lower concentration due to saturation of the capture antibody and the antibody conju- gate by the high concentration of analyte (Akamatsu et al., 2006). A similar effect can be seen in both qualitative and quantitative hCG assays due the presence of hCGβ, a sub- unit of the analyte (Grenache et al., 2010; Gronowski et al., 2009). In some assays that do not detect hCGβ, falsely decreased results can also occur because the competing hCGβ molecule saturates just one of the antibodies (Grenache et al., 2010). Strategies to Identify Potential Cases of Assay Interference There have been many strategies proposed for the detec- tion of assay interference within the context of the clinical laboratory. Many of these methods are technical tests, measurements, or treatments that can be performed in the laboratory, but a powerful tool for discovering and con- firming the presence of assay interference is recognition of the clinical context of the test. Indeed, in many cases of medical misadventure and poor patient outcome, the clini- cian ordering the test does not initially believe the patient’s test result. But because they are not aware of immunoassay interference, they repeat the erroneous test enough times to convince themselves of the test result’s clinical signifi- cance. Sometimes, the tragic consequence is unnecessary surgery, chemotherapy, and/or sterilization. In the ideal world, the clinician faced with a test result that does not fit with the patient’s presentation will con- sult the laboratory. Whenever a clinician (or patient) contacts the laboratory with doubt about an immunoas- say result, this is an opportunity to consider the possibil- ity of assay interference. If this opportunity is missed, then there is little use for knowing the myriad of meth- ods available for evaluating and proving immunoassay interference. In addition to increasing awareness of immunoassay interference among clinical colleagues, the laboratory can identify potential cases of interference by examining the consistency of test results with other laboratory values and evaluating the probability of the test result in the context of the prevalence of disease (Bayesian analysis). CLINICAL AWARENESS There is not always a proper venue to properly educate clinical colleagues to the possibility of immunoassay inter- ference. The usual opportunities to educate clinicians within the context of a hospital laboratory may be through participation in teaching rounds, clinical conferences, lab- oratory newsletters, and notifications through electronic medical record and laboratory test ordering systems. Another opportunity is to educate colleagues in anatomic/ surgical pathology that may interact regularly and closely with clinicians on specific cases. In our experience, this is particularly effective in the setting of oncology conferences where immunoassays for tumor markers are routinely dis- cussed. A surgical pathologist that is present to discuss his- tologic findings may also seize the opportunity to mention the possibility of immunoassay interference when the discus- sion arises of an unexpected serum tumor marker result. EXAMINATION OF PLAUSIBILITY OF TEST RESULTS A comprehensive interpretation of immunoassay results can be performed in the context of additional laboratory testing or in the context of statistical probability based on disease prevalence. These types of tools can be used in quality assurance audits or can be potentially automated within the laboratory information system. Regarding the interpretation of test results in the con- text of all available laboratory data, a recent study of para- thyroid hormone (PTH) identified potential cases of heterophile antibodies by excluding the cases that had a “clinically plausible reason” as determined by laboratory values (Cavalier et al., 2008). This study interpreted PTH in combination with serum 25-hydroxyvitamin D (25VTD), ionized calcium, and estimated glomerular fil- tration rate (eGFR). A rule was set that flagged an elevated PTH as suspicious when 25VTD, ionized calcium, and eGFR from the same patient were all within their respec- tive normal reference ranges. Based on this rule, 9% of samples with elevated PTH were deemed suspicious and were treated in heterophile antibody blocking tubes (Scantibodies, Santee, CA). Of these suspicious samples, 40% had a decrease in PTH after treatment with blocking tubes; half of these samples with a decrease had a final value that fell into the normal reference range. The remaining 60% of suspicious samples (unchanged by het- erophile blocking) were then examined for the presence of RF; if the sample was RF positive, then the sample was treated with an RF precipitating reagent (IBL, Hamburg, Germany), and this revealed that 24% of heterophile negative samples had interference attributable to RF. This method of identifying suspicious samples has the potential for wider implementation in laboratories with sophisticated laboratory information systems. Indeed, as a matter of routine quality assurance, rules can be written into information systems to flag suspicious immunoassay results that are not plausible based on the context of other laboratory test results. In addition to PTH, thyroid function testing and other hormone immunoassays are usually part of a multi-analyte or multiparameter analysis and would be amenable to this identification strategy. Based on the identification of suspicious cases, a more
  8. 8. 410 The Immunoassay Handbook intensive chart review or laboratory investigation could be conducted. Another powerful tool in the identification of immuno- assay interference is to determine the probability of a test result based on the disease prevalence. Previous studies have examined the utility of statistical methods in uncover- ing laboratory errors (Le et al., 2011; Oosterhuis et al., 2000). A recent study applied the statistical method of Bayesian analysis to identify immunoassay interference (Ismail and Ismail, 2011). The authors of this study dem- onstrated the potential of Bayesian analysis in identifying interference with various analytes including TSH, PSA, hCG, and troponin. In the example of PSA, the starting point of analysis is the recognition of prostate cancer prev- alence of approximately 1 in 5000 (0.02%) among asymp- tomatic 50-year-old men. Using the 0.4% false-positive rate of immunoassays reported in the literature, the calcu- lated probability of a false-positive test is approximately 95%. With increasing age, the prevalence of disease increases, and therefore, the false-positive rate decreases. Thus, elevations of PSA at a younger age are more suspi- cious than at an older age. In the example of TSH, sub- clinical hypothyroidism has a prevalence of 1% in young adults and children compared to a prevalence of 17% in elderly females. Again, assuming a false-positive immuno- assay interference of 0.4%, a raised TSH in elderly female patients would be false in 2% of cases compared to 30% of cases in young adults or children. Reexamining all immunoassay results in a clinical labo- ratory is not practical, but developing a targeted approach that can be automated within the laboratory information system may provide an additional level of security in the identification of immunoassay interference. Strategies to Prove the Presence of Interference Once a potential interference has been identified, there are several methods of proving that the test result is due to interference (Sturgeon and Viljoen, 2011). LINEAR DILUTION The simplest and first check for an immunoassay assay result that is outside the reference range is to perform a linear dilution. Immunoassay interferences including heterophile antibodies do not typically dilute linearly. Dilution ratios of 1:5 and 1:10 are commonly used for such studies, and Table 5 (Landau-Levine et al., 1999; Pan and Wang, 2011; Zhu et al., 2008) shows some examples of results of dilution studies for specimens containing an interferent. TEST BY ANOTHER METHOD Immunoassay interferences can be specific to the antibody clones used in the assay. When interference is suspected, the sample should be tested by other methods. The most important consideration is that the alternative method must use different antibody clones. It is common to find immunoassay reagents from different manufacturers that use the same antibody clones for a given analyte. Further- more, if the sample is sent to a third party laboratory to be tested, the specific method and antibody clones used should once again be verified. Since urine is free of immunoglobulins, examining urine for the analyte of interest may be more reliable. For TABLE 5 Examples of Dilution and Blocking Studies for Specimens Containing Interferents: Patient A: Positive Interference due to HAMA. Patient B: Positive Interference and Use of HBT. Patient C: Positive Interference and Use of IgG Mixture as Blocker Dilution Factor Result Corrected for Dilution Comments Patient A (Pan and Wang, 2011) BNP assay (pg/mL) Untreated 1 1551 Does not dilute linearly. HAMA assay was positive (40.5ug/L) thus confirming HAMA as the interferent Untreated 2 1061 Untreated 5 247 Untreated 10 31.9 Untreated 100 2.56 Patient B (Zhu et al., 2008) cTnI (µg/L) Untreated 1 19.99 Initial result differed from TnI test performed on patient by another method Untreated Diluted 1.46 Does not dilute linearly (93% decrease) Treated in a HBT 1 0.03 Reduction in test value after incubation in HBT indicates a heterophile antibody interference Patient C (Landau-Levine et al., 1999) TSH (mIU/L) Untreated 1 9.9 Initial result differed from TSH test performed by another laboratory +Mouse and goat IgG 1 1.8 Reduction in test value after incubation with IgG mixture indicates an interference, confirmed as HAMA by assay (44 µg/L)
  9. 9. 411CHAPTER 5.3 Interferences in Immunoassay example, with β-hCG, the serum test is susceptible to interference, but the urine is not. This property of urine is the basis of a strategy recommended by American Congress of Obstetricians and Gynecologists (ACOG) in the case of false-positive serum hCG test results (ACOG. Committee, 2002). TREAT THE SAMPLE TO REMOVE INTERFERENTS There are several chemical treatments and multiple com- mercially manufactured reagents available to remove or block heterophile antibodies from a sample. Strategies that involve relatively mild conditions to remove interferents include PEG precipitation and exposure to immobilized Protein A or Protein G. For more stable analytes such as CEA, acid extraction or heat inactivation can be used to remove interferents (Primus et al., 1988). However, a par- ticularly convenient method is to use a blocking tube— e.g., a Heterophilic Blocking Tube (HBT) or a Nonspecific Antibody Blocking Tube (NABT) (Scantibodies Labora- tories, Inc. Santee, CA) (http://www.scantibodies.com/ blockers.html). A sample of the specimen is incubated in the tube and the reagents contained in a lyophilized pellet in the tube bind to any interferents in the sample. This type of pretreatment procedure is only intended to confirm the original assay result or show the result to be incorrect due to the presence of an interferent—it cannot be used to gener- ate a reportable result. Another nonspecific sample pretreat- ment procedure is to add animal immunoglobulin or nonimmune serum. This may not always be successful, and in one case, blocking of the interference required the spe- cific monoclonal antibody that had been infused into the patient and also required prolonged incubation with high concentrations of the antibody (Kricka et al., 1990). There is also a range of specially formulated blockers from a number of commercial sources (see BLOCKERS AND TEST PANELS). Measures to Prevent Immunoassay Interference and Existing Regulatory Guidelines The time to identify an immunoassay’s susceptibility to interference is during the development and validation of the test prior to clinical use. As well as checking the immu- noassay reagents for interference, preanalytical variables should also be examined. These variables may include drugs the patient may be taking that will be present in the serum, the type of anticoagulant the sample will be col- lected with, and finally the type of tube that is used for collection (see BLOOD COLLECTION TUBES). BLOCKERS AND TEST PANELS Many commercial assays include blocking agents, and there are panels of samples that are positive for interferent (e.g., heterophile antibodies, RF) that can be used to assess and validate the effectiveness of the blocking agent chosen for an assay. As exemplified by a recent study of a cytokine immuno- assay targeted at patients with autoimmune disease, when targeting a patient population with a high potential for interference, multiple heterophile antibody blocking reagents can be surveyed to find the best reagent to elimi- nate interference (DeForge et al., 2010). In addition to identifying a blocking reagent during the test validation, the various blocking reagents can be examined to see if they are compatible with the test (e.g., examine whether the blocking reagents cause interference). Different formulations of blocking agent are available from several companies including Scantibodies Labora- tories, Inc. (Santee, CA), Bioreclamation, LLC (Hicks- ville, NY), Millipore (Danvers, MA), IBL International (Hamburg, Germany), and Meridian Life Science Inc. (Cincinnati, OH). REGULATIONS AND STANDARDS The general concept of analytical interference in the clini- cal laboratory has been well recognized for several decades. For immunoassays, laboratories have been compelled by federal law (CLIA ‘88) to check for common interferences such as lipemia, hemolysis, and icterus during the validation of a new test (Medicare, 1992). Immunoassay interference must also be determined by the clinical assay manufacturer and is a standard requirement for the clearance or approval of a new immunoassay by the U.S. Food and Drug Admin- istration. However, there is minimal regulatory guidance on the appropriate approach to interference from endoge- nous antibodies. There is a recent comprehensive CLSI document, which is recognized by the FDA as a complete standard (Clin. Lab. Stds. Inst., 2008). The CLIA ‘88 requirements for assessing the effect of interference on clinical laboratory tests are extended by organizations with deemed status for the accreditation and inspection of clinical laboratories in the United States (e.g., College of American Pathologists, Joint Commission). Clinical Consequences of Immunoassay Interference The following clinical cases illustrate some dire conse- quences of immunoassay interferences. RUFER CASE There are now many documented case reports and case series of clinically significant immunoassay interference. However, one case in particular gained tremendous media attention in the United States because of the clinical con- sequences, timeline to resolution, and eventual economic judgment through the judicial system. Due to a falsely positive serum hCG, Jennifer Rufer was subjected to doz- ens of clinical tests, radiological imaging, multiple doses of chemotherapy, lung surgery, and hysterectomy (Rufer, 2003, 2005). Ms Rufer initially presented with abdominal pain and vaginal bleeding and was diagnosed as having an ectopic pregnancy by an obstetrician–gynecologist. She had an elevated serum hCG determined by a commercial
  10. 10. 412 The Immunoassay Handbook laboratory; this was consistent with ectopic pregnancy, and the patient was treated with low-dose chemotherapy. However, the hCG did not decrease with treatment and so she was referred to a gynecologic oncologist at a Univer- sity hospital. She continued to have a persistently elevated serum hCG determined by the University hospital labora- tory; this resulted in her being diagnosed with gestational trophoblastic disease and she was subjected to higher doses of chemotherapy, hysterectomy, and lung surgery for sus- picion of metastatic disease. Unfortunately, both the commercial laboratory and University hospital were using hCG assays from the same manufacturer. The laboratory director at the University hospital conducted an extensive investigation of the test by examining different batches of reagent and performing dilution studies on the patient’s sample. Ms Rufer’s sample was the only sample, out of 50 that were tested, that failed the dilution analysis. This result was reported to the man- ufacturer, but the manufacturer did not subsequently respond to the clinical laboratory’s request for assistance in interpretation. The possibility of falsely elevated hCG due to interference was not well known in the medical lit- erature at that time. The package insert from the manufac- turer stated the following limitations: 1. For diagnostic purposes, the hCG results should be used in conjunction with other data: e.g., symptoms, results of other tests, clinical impressions. 2. Elevated hCG levels have been associated with some abnormal physiological states and should be considered if consistent with clinical evidence. 3. Specimens from patients who have received prepa- rations of mouse monoclonal antibodies for diagno- sis or therapy may contain HAMAs. Such specimens may show either falsely elevated or depressed values when tested with assay kits which employ mouse monoclonal antibodies. 4. Infrequently, hCG levels may appear consistently elevated due to the presence of heterophilic anti- bodies or to nonspecific protein binding. If the hCG level is inconsistent with clinical evidence, results should be confirmed by an alternate hCG method. Finally, the University laboratory sent the sample to two other clinical laboratories: one that used a different manu- facturer found a normal result; the second used the same manufacturer as the University laboratory and similarly found an elevated level. The manufacturer performed its own internal assessment and initially concluded that the assay was performing as intended. The University hospital physicians eventually determined that Ms Rufer’s elevated serum hCG was due to interference and her medical treat- ments had all been unnecessary, Ms Rufer sued both the University hospital and the manufacturer of the hCG test. The University hospital was sued for malpractice, and the manufacturer of the test was sued for product liability. The product liability was because it was believed that the man- ufacturer had prior knowledge and had not warned physi- cians that their test could produce false-positive results leading to misdiagnosis and unnecessary treatment for gestational trophoblastic disease. The package insert was found to be inadequate because it did not specify that het- erophile elevations of the assay could actually be false, phantom, or otherwise analytically in doubt. Furthermore, it was found that the manufacturer had received over 40 complaints of false-positive results including multiple cases similar to Ms Rufer’s with unnecessary chemother- apy and surgery. In the fall of the same year that Ms Rufer was initially diagnosed, the manufacturer had drafted, but never sent out, a general letter to physicians recommend- ing a urine test to confirm serum hCG results that were in doubt. A jury trial awarded Ms Rufer and her husband $16 million in damages with 50% of the fault placed on the University hospital. The Rufer case has created heightened awareness among clinicians, laboratorians, and test manufacturers of immu- noassay interference by endogenous antibodies. Some important lessons include: 1. Immunoassay should not be used as a sole diagnos- tic tool; it must be used in conjunction with other clinical findings, imaging studies, or laboratory values. 2. False immunoassay results can have catastrophic clinical consequences. 3. Concerns or doubts by clinicians of test results must be treated with the utmost respect and be investigated. 4. Laboratories cannot place the responsibility of investigation solely on the test manufacturer. 5. Laboratories in the United States potentially hold equal liability with the manufacturers of the tests they perform. MALE UROLOGIC ONCOLOGY PATIENTS WITH HETEROPHILE INTERFERENCE In a series of cases reported in 2000, it was observed that serum markers of testicular cancer can be falsely elevated in the absence of active disease (Morris and Bosl, 2000); however, this series did not identify the mechanism of false positivity, and heterophile antibodies were not described as a possible etiology. Subsequently, there have been sev- eral cases of heterophile antibodies with clinical signifi- cance in urologic oncology patients. In one case, a 39-year-old male with stage IIB metastatic malignant germ cell tumor (GCT) and elevated serum hCG underwent orchiectomy with three cycles of combi- nation chemotherapy (Ballieux et al., 2008). Serum hCG initially normalized, but then 2 months later, hCG was elevated. Patient underwent another operation for retro- peritoneal lymph node detection which was negative for tumor. Serum hCG became further elevated and a chest CT-scan revealed enlarged mediastinal lymph nodes. The patient was then treated with second and third-line che- motherapy which did not normalize his serum hCG. Reanalysis revealed that the serum samples prior to the first round of chemotherapy had confirmed elevated serum hCG; however, all subsequent serum hCG elevations were found to be false positives. Additional cases of heterophile interference in male patients include a 44-year-old man with an orchiectomy for low-stage nonseminomatous GCT who has a persistently elevated hCG after chemotherapy (Gallagher et al., 2010), and a 43-year-old man with orchiectomy for low-stage
  11. 11. 413CHAPTER 5.3 Interferences in Immunoassay seminomatous GCT who had a falsely elevated hCG at his routine 5-year follow-up (Trojan et al., 2004). THE ANIMALS WE LOVE Human Anti-rabbit Antibody (HARA) A bad outcome from anti-rabbit antibodies is illustrated by a case of a woman in her late twenties who presented with infertility and amenorrhea (Berglund and Holmberg, 1989). High serum FSH values were noted, and this led to a series of unnecessary diagnostic procedures, including laparos- copy, laparotomy, and an ovarian biopsy. After a few months, the patient’s regular menstrual cycle resumed spontaneously. However, her FSH levels remained elevated. Reanalysis of her samples with a goat antibody-based assay gave normal values for FSH. A better outcome from anti-rabbit antibodies is illus- trated by a case of a 52-year-old woman who was referred for further investigation because of a 9-year history of per- sistently raised fasting concentrations of gut hormones. Irritable bowel syndrome had been diagnosed 16 year pre- viously (Park et al., 2003). Previous investigations included computed tomography scan of the abdomen, magnetic resonance imaging of the pancreas, and an octreotide scan. All of these imaging studies were normal; therefore, the abnormal blood test results were attributed to hyperplasia of pancreatic islet cells. The only drug she was receiving was estrogen from an estrogen implant. Two yearly magnetic resonance imaging studies were normal, but there was a suggestion of hyperplasia of the pancreatic islet cells. On referral, many of the patient’s fasting gut hormone concentrations were still elevated (vasoactive intestinal polypeptide 120pmol/L [normal, <30], pancreatic poly- peptide 72pmol/L [<300], gastrin 125pmol/L [<30], glucagon 63pmol/L [<50], somatostatin 103pmol/L [<150], and neurotensin 101pmol/L [<100]). Imaging of the abdomen and an octreotide scan were normal, and the patient was scheduled for pancreatic angiography with cal- cium stimulation to ascertain whether she had abnormal functioning islet cells. Because it is unusual for a neuroendocrine tumor to secrete more than one hormone, the possibility of interfer- ing antibodies was considered for this patient. A more detailed clinical history from the patient revealed that she and her husband had kept large numbers of pet rabbits (as many as 80 at one time) and that she had a presumed rabbit induced allergic rhinitis. A further link to rabbits was that her husband was an officer of the British Rabbit Council. The RIAs used to measure gut hormones in this patient were all based on rabbit antibodies. The gastrin assay was selected for blocking studies, which were performed by adding small concentrations of nonimmune rabbit serum to the gastrin assay buffer. The original gastrin result was elevated at 95pmol/L (no rabbit serum added). Addition of 0.5 and 1% of rabbit serum reduced the gastrin result to <20pmol/L, which is in the normal range (<30pmol/L). The angiography was canceled, and the patient was dis- charged with the diagnosis of presumed irritable bowel syndrome with heterophilic (rabbit) antibodies interfering with the various gut hormone assays. Human Anti-goat Antibody (HAGA) (Alvarez and Scott, 1993) An 84-year-old woman had a discordant, elevated creatine kinase (CK) isoenzyme by immunoassay (Stratus CK-MB assay result, 12–15fg/L) and by electrophoresis (>95% MM isoenzyme, no detectable MB). Mouse IgG was added to test for HAMA and had no effect, indicating that the sam- ple probably did not contain HAMA. However, the addi- tion of normal goat serum reduced the measured CK-MB to 1.7fg/L. This suggested that a human anti-goat antibody was the most likely cause of the interference. The Stratus CK-MB assay includes goat IgG as a component of the anti-CK-MB-alkaline phosphatase conjugate reagent. When a CK-MB conjugate that did not contain goat IgG was utilized, the interference similarly disappeared. It was surmised that the interfering antibody reacted with the goat IgG, and the resulting complex caused unreacted conjugate to be retained in the central measurement zone of the radial diffusion device used in the Stratus immunoassay. THE DRUGS WE TAKE (PARIKH ET AL., 2010) A 35-year-old man with end-stage renal disease received a cadaveric renal transplant. According to protocol, on the day of transplantation, his immune suppression was initi- ated by rabbit-ATG; on the day after transplantation, he continued immunosuppression by mycophenolate mofetil, prednisone, and tacrolimus. Twelve hours after his first 1mg dose of tacrolimus, his whole-blood concentration was determined to be 24.4ng/mL by a Siemens Dimen- sion® RxL immunoassay. This tacrolimus level was elevated since the target whole-blood concentration is 5–15ng/mL after transplantation. The patient continued to do well clinically without signs or symptoms of drug toxicity or rejection and had repeat measurements of tacrolimus. Tacrolimus concentrations remained elevated at 24.0 and 23.6ng/mL on the third and fourth days after transplantation, respectively. The patient was assessed for RF, but this was negative. After obtaining the elevated tacrolimus level on the fourth day of transplantation, the clinical laboratory had the timely insight to send a blood sample to a reference laboratory for tacrolimus measure- ment by liquid chromatography–tandem mass spectrome- try (LC–MS/MS). The reference laboratory determined the tacrolimus level to be <2ng/mL. Therefore, the patient’s tacrolimus dosage was increased to 2mg twice a day. The patient continued to have tacrolimus levels per- formed on LC–MS/MS, and these levels began to fall within the therapeutic range. The patient’s sera were investigated for interference by HAMA, but treatment by HBR (Scantibodies Lab. Inc) or irrelevant murine monoclonal antibodies did not remove the interference. The sera were then investigated for anti- bodies directed against the beta-galactosidase label by examining the sera’s ability to interfere in the Siemens RxL cyclosporine A immunoassay that utilizes the identi- cal beta-galactosidase conjugate. The patient’s sera dem- onstrated no interference on the Siemens cyclosporine assay. Subsequent investigation revealed the interfering antibody to be directed against an epitope generated by
  12. 12. 414 The Immunoassay Handbook the conjugation of the anti-tacrolimus monoclonal anti- body to the beta-galactosidase reporter. This case illustrates how interfering antibodies can defy the standard methods of investigating for assay interfer- ence by endogenous antibodies. The authors of this case report suggest a potential solution for uncovering interfer- ence in immunoassays used for therapeutic drug monitor- ing. They recommend performing the first measurement of the drug of interest before initiating immunosuppres- sion. Thus, the false-positive detection by interference can be immediately identified. Summary Immunoassay is a powerful technology, however, it should be remembered that it is an estimate for biological pro- cesses. Caution should be taken by treating physicians when the result of an immunoassay does not fit the overall clinical picture of a patient. As laboratorians, our role is to be vigilant to the concerns of clinicians and patients. 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